Plant cell culture expression systems have several advantages over bacterial, yeast, or Baculoviral expression systems. Bacteria do not, and yeasts only limitedly, carry out post-translational modifications of the recombinant eukaryotic proteins they express. Such modifications are often necessary for the proper function of proteins. Baculovirus is a potent transformation vehicle for higher eukaryotes, and in general, proteins expressed by Baculoviral expression systems are properply modified. However, the cost for culturing Baculovirus is high. In addition, the host cells are eventually lysed by Baculovirus, resulting in the contamination of the expressed recombinant protein by thousands of host proteins released into the culture medium. Consequently, purification of the expressed recombinant protein may be rendered difficult.
Plant cells, on the other hand, are higher eukaryotic cells and thus able to perform sophisticated post-translational protein modifications of expressed eukaryotic proteins. Plant cells can also secrete the expressed proteins into culture media, making the purification of the proteins easier. Media for plant cell culture mainly contain salts and vitamins, and no serum supplement is required. Thus, the media cost much less than those used to culture insect cells which are used for the Baculovirus transfection.
Plant cell cultures are a potential commercial source of medicines, dyes, enzymes, flavoring agents and aromatic oils. Plant cell culture production of such components are sought when (1) they are naturally produced by plants in small quantities, or in fleeting or unharvestable developmental stages of the plants' life cycle; (2) they are produced by plants which are not amenable to agriculture or are native to vanishing or inaccessible environments; and (3) they cannot be satisfactorily synthesized in vitro.
Attempts to produce products by plant cell culture, however, are often commercially unsuccessful due to such factors as insufficient production or secretion of the desired product, poor cell growth, and difficulties in maintaining the appropriate cell type in culture.
The .alpha.-amylase expression system of rice callus has features that make it of potential use in plant cell fermentation technology. These features include high and sustained levels of expression, expression irrespective of either the tissue origin of the cell culture or tissue formation in the culture, and the ability to secrete the recombinant protein products.
.alpha.-amylases are the major amylolytic enzymes for hydrolysis of starch stored in the endosperm during germination of cereal grains. These enzymes catalyze the hydrolysis of .alpha.-1,4 linked glucose polymers. During the initial germinating period, cells in the aleurone layer of seeds synthesize .alpha.-amylases. Together with a-glucosidase and enzymes restricting dextrinase, the .alpha.-amylases are secreted into the endosperm and hydrolyze starch to form glucose and maltose, providing the nutrients needed for the growth of the germ (Rogers et al., J. Biol. Chem., 259:12234-12240, 1984; Rogers, J. Biol. Chem., 260:3731-3738, 1985).
Currently, 7 .alpha.-amylase cDNA's and 9 .alpha.-amylase genomic DNA groups have been cloned in barley (Chandler et al., Plant. Mol. Biol., 3:401-418, 1984; Deikman et al., Plant Physiol., 78:192-198, 1985; Krushseed et al., J. Biol. Chem., 263:18953-18960, 1988; Knox et al., Plant Molecular Biology, 9:3-17, 1987). The .alpha.-amylase genes of wheat are grouped into .alpha.-Amy1, .alpha.-Amy2, and .alpha.-Amy3. The proteins encoded by .alpha.-Amy1 and .alpha.-Amy2 have high and low isoelectric points, respectively, and these two gene groups each include more than 10 genes that are expressed in germinating seeds. The .alpha.-Amy3 gene group includes 3-4 genes, and they are expressed in immature seeds (Baulcombe et al., Mol Gen. Genet., 209:33-40, 1987).
Rice .alpha.-amylase isozymes are encoded by at least nine genes (Thomas et al., Plant Physiol., 106:1235-1239, 1994). Expression of .alpha.-amylase genes in rice has been found to be under different modes of tissue-specific regulation: in the embryo of germinating seeds and in suspension cultured cells, expression is activated by sugar deprivation and repressed by sugar provision (Karrer et al., Plant J., 2:517-523, 1992; Yu et al., J. Biol. Chem., 266:21131-21137, 1991; Yu et al., Gene, 122:247-253, 1992; and Yu et al., Plant Mol. Biol., 30:1277-1289, 1996). In the endosperm of germinating seeds, expression is activated by gibberellic acid and repressed by abscisic acid and osmotic stress (Itoh et al., Plant Physiol., 107:25-31, 1995; and Yu et al., Plant Mol. Biol., 30:1277-1289, 1996). Studies with rice suspension cells have shown that .alpha.-amylase expression, carbohydrate metabolism, and vascular autophagy are coordinately regulated by sucrose levels in the medium (Chen et al., Plant J., 6:625-636, 1994). Both the transcription rate and mRNA stability of .alpha.-amylase gene in cells increases in response to sucrose depletion in the culture medium (Sheu et al., Plant J., 5:655-664, 1994). Studies using transgenic rice carrying .beta.-glucuronidase (GUS) gene under the transcriptional control of an .alpha.-amylase gene promoter proved that the regulation of .alpha.-amylase gene expression by sugars involves a transcriptional control mechanism (Chan et al., Plant Mol. Biol., 22:491-506, 1993; Chan et al., J. Biol. Chem., 269:17635-17641, 1994; and Huang et al., Plant Mol. Biol., 23:737-747, 1993). Sugar-dependent repression of .alpha.-amylase gene expression has also been observed in Aspergillus oryzae (Tonomura et al., Agric. Biol. Chem., 25:1-6, 1961) and Drosophila melanogaster (Benkel et al., Proc. Natl. Acad. Sci. USA, 84:1337-1339, 1987), and the mechanism was shown to involve transcriptional control (Magoulas et al., Genetics, 134:507-515, 1993; and Tsuchiya et al., Biosci. Biotech. Biochem., 56:1849-1853, 1992).
The synthesis of .alpha.-amylases and levels of their mRNA are greatly induced under sucrose starvation. An increase of .alpha.-amylase synthesis is assumed to accelerate hydrolysis of cellular starch as an energy source when exogenous carbon source is depleted. Under normal growth condition with an adequate supply of sugar, .alpha.-amylase is subjected to metabolite repression. It has been further observed that .alpha.-amylases synthesized by cultured rice cells are secreted into the culture medium and can account for about 15-20% of the total proteins present in the medium during periods of sugar depletion.
By using .alpha.-amylase gene-specific DNA fragments and nuclear run-on transcription analysis, transcription of eight .alpha.-amylase genes has been shown to increase in response to sucrose starvation (Sheu et al., J. Biol. Chem., 271:26998-27004, 1996). A positive correlation between the transcription rates and the steady-state mRNA levels suggests that transcription regulation plays an important role in the differential expression of individual .alpha.-amylase genes.
U.S. Pat. No. 5,460,952 discloses a gene expression system utilizing the transcriptional regulation characteristics of the .alpha.-amylase gene promoter regions. In this system, an .alpha.-amylase promoter controls the expression of foreign genes in transformed plant cells as well as the secretion of the foreign gene products into the medium.